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Neurotransmitter Transport Research

Communication between neurons is achieved through release of small molecules called neurotransmitters from the pre-synaptic neuron. This chemical signal diffuses across a synaptic cleft to interact with specific receptors on the post-synaptic neuron in order to elicit a biochemical response. The biogenic amines dopamine, norepinephrine and serotonin are neurotransmitters that have been shown to mediate physiological processes such as reward and addiction, movement and mental health.

For the biogenic amines, synaptic transmission is terminated by integral membrane proteins, called neurotransmitter transporters, located on the pre-synaptic terminal. These transporters utilize the asymmetric distribution of Na+ and Cl- across the plasma membrane to catalyze the thermodynamically unfavorable movement of neurotransmitter back into the pre-synaptic neuron for replenishment of neurotransmitter stores and subsequent neural firing. Because transport of substrate is coupled to ion transport (symport), these secondary active transporters are known as neurotransmitter sodium symporters (NSS). Dysfunction of NSS leads to a variety of disorders such as orthostatic intolerance and depression. Not surprisingly, these proteins are the primary pharmacological targets of antidepressants such as fluoxetine (Prozac) which is specific for the serotonin transporter. Furthermore, studies have shown that these transporters directly interact with drugs of abuse, cocaine and amphetamine.

Although functional studies have provided insights into the mechanism of substrate transport, detailed structural analysis cannot be addressed due to the lack of large-scale heterologous expression systems generating material suitable for high resolution structure determination. However, homologous prokaryotic transporters can be obtained in large quantities, potentially providing the means to unveil the conformational changes associated with the transport process. Recently, Yamashita and colleagues reported the crystal structure of a bacterial homologue for neurotransmitter transporters, the sodium-dependent leucine transporter (LeuT) from Aquifex aeolicus.

In the crystal structure of LeuT, the two coordinated Na+ and bound leucine are occluded from both the extracellular and intracellular domains, yielding limited information in terms of a permeation pathway. According to the alternating access model of transport, this structure may be interpreted as a transient intermediate state. Some persistent questions remain: How does Na+ coordination structurally translate into energy input? Does the binding of substrate induce a conformational change consistent with closure of the extracellular domain? In collaboration with the Javitch group from Columbia University, we intend to address these issues using a combined computational modeling and site directed spin labeling/EPR approach.

LeuTAlternating Access Model
  Crystal structure and proposed conformational changes of LeuT. In the crystal structure, two Na+ and leucine are centrally bound and occluded. Although only a monomer is shown, LeuT crystallizes as a dimer with an interface at transmembrane helices 9 and 12. According to the alternating access model, the substrate binding site will be alternately exposed from the extracellular milieu (outward facing) to the cytoplasm (inward facing).
Current Researchers
Derek Paul Claxton, Graduate Student. Since little is known about the molecular mechanism, my work seeks to determine the conformational changes that occur during binding and transport of substrate using the prokaryotic homolog leucine transporter (LeuT).
Recent Publications